Fiber-coupled multiplexed confocal microscope

ABSTRACT

A confocal microscope system that is inherently fiberoptic compatible is described which has line scanning aided image formation. An incoherent fiberoptic bundle maps a line illumination pattern into a dispersible group of separate sources, and then remaps this confocally selected remitted light to the original line. Fibers, not confocal with the illumination, carry light to be rejected from the image back on itself upon double passing, while separate fibers carry light from non-confocal sample planes. The transformation allows efficient rejection of unwanted photons at a slit aperture. The fiber bundle and an objective lens provide a flexible probe for imaging internal tissue for pathological examination on a cellular level.

[0001] This application claims the priority benefit of U.S. ProvisionalApplication No. 60/215,154, filed Jun. 30, 2000, which is hereinincorporated by reference.

DESCRIPTION

[0002] The present invention relates to confocal microscopy, and relatesparticularly to confocal microscope having a parallel scanning systemcompatible with fiberoptics. The invention provides a remote probe forconfocal imaging of tissue at locations within a body, such as commonlydone with endoscopes, and thus the invention provides the advantages ofconfocal microscopy in biomedical applications by enabling access todistant and inconvenient regions.

[0003] The invention uses fiber optics, but in a way to providesignificant improvement over confocal microscopes using fibers that havebeen reported, such as in which a single fiber serves as the source andthe detector pinhole.

[0004] A line scanning confocal microscope with slit aperture detectioncan be regarded as a form of multifocal illumination and paralleldetection, where all the foci line up in one direction. Image formationrequires only slow scanning (at 25 or 30 Hz for video rate) in thesecond direction. This approach is particularly attractive because ofits simplicity and high optical throughput. The major drawback of theline scanning approach is its relative poor rejection of unwantedphotons compared to the pinhole system.

[0005] Briefly, the present invention provides a confocal microscope inwhich the region of interest (as for example a tissue ex-vivo, orin-vivo as inside a body cavity) is illuminated via a fiber opticbundle, where the spatial arrangement of fibers at one end of the bundleis different from that at the other end. Such a bundle is called anincoherent fiber optic bundle. A bundle in which the spatial arrangementof fibers is maintained is a coherent fiber bundle. The microscope mayhave means for scanning a laser beam from a slit providing an aperturein one direction across, as with a slit scanning microscope, but hastrue two-dimensional confocality because of encoding the slit with anincoherent fiber bundle. The incoherent bundle effectively multiplexesand demultiplexes the light, respectively, incident on and remitted fromthe region of interest. An important advantage of this microscope isthat it can operate at the distal end of a fiber bundle and cantherefore provide a probe on a flexible, small diameter member where itcan be implemented on or as an endoscope or catheter.

[0006] It is a feature of the invention to provide an improved fiberoptic confocal microscope where the fiber effects a parallel scanningmechanism that is inherently fiberoptic compatible and that retains thesimplicity of the line scanning confocal microscope, while improving onits axial sectioning resolution. The improvement is provided by using anincoherent fiber bundle. In the bundle, the input and output fibers maybe randomly arranged, although other, specific mapping can also beemployed to obtain incoherent coupling via the fiber. A line sourceilluminates the proximal (P) end of the fiber bundle, which transformsthe line input into disperse array of fibers at the distal (D) end,spread out over the whole bundle. This set of disperse spots is imagedonto the sample by an objective lens. Remitted light (fluorescence orback scattered) from the sample is collected and imaged by the sameobjective lens back onto the fiber endface (D), from a region in thesample being viewed, which is exactly in focus, the remitted light isimaged onto the same set of fibers which carried the illumination light,and transformed back into a line at (P). For an unwanted (orout-of-focus) sample plane, each illuminated fiber at D will produce, onthe return, a smeared-out spot, which covers not just the original fiberbut a group of surrounding fibers. For spots illuminated indirectly,such as by scattered light, the same is true. Back at Plane P, thefibers which carry the “smeared-out” photons do not reassemble into aline but are spread out dispersely over the bundle. A slit aperture,placed at a plane conjugate to P, allows only light (photons) from thein-focus plane, and intentionally illuminated spots, to pass through thedetector, while rejecting most of the unwanted photons. The detectorprovides signals representing scan lines in the plane from which a 2-Dimage at that plane can be constructed.

[0007] The invention will become more apparent from a reading of thefollowing specification in connection with the accompanying drawingswhich, briefly described, are as follows:

[0008]FIG. 1A is a view of an end of a fiber optic bundle which showsonly a center fiber thereof that is illuminated.

[0009]FIG. 1B is a view in a plane, D, across the distal end of thefiber optic bundle of FIG. 1A when that bundle represents a coherentfiber optic bundle.

[0010]FIG. 1C is a view in a plane, P, across the proximal end of thebundle, and showing the location of a slit aperture associated with thebundle of FIG. 1B.

[0011]FIG. 1D is a view in the plane, D, for the fiber optic bundle ofFIG. 1A when that bundle is an incoherent fiber optic bundle accordingto the present invention.

[0012]FIGS. 2A, 2B and 2C are schematic diagrams of differentembodiments of confocal microscope systems embodying the invention.

[0013]FIGS. 3A and 3B are exemplary images of the P and D ends which canbe taken with the microscope of FIGS. 2A, 2B or 2C.

[0014]FIG. 4 are plots showing the intensity variation of the lightthrough a confocal slit aperture (direction z) showing thediscriminating effect of the incoherent bundle by the curve having thesolid dots, and without the slit aperture in the curve having the opendots.

[0015] Consider FIGS. 1A to 1D and that, for clarity, a single fiber atthe center of the bundle is illuminated (FIG. 1A). Return light from anon-confocal point is collected by the objective lens and forms andextended spot at plane D, filling a group of fibers surrounding thecentral fiber (FIG. 1B). If the fiber bundle is coherent, this group offibers will emerge at plane P with a similar pattern to that in FIG. 1Bbut surrounding the fiber of FIG. 1A. The planes P and D are at theproximal and distal ends of the bundle and are perpendicular to theoptical axis (see FIGS. 2A-C).

[0016] A confocal slit aperture placed at a plane conjugate to P andaligned with the central fiber will reject much of the unwanted light(FIG. 1C). However, a significant fraction of the unwanted light will gothrough the slit. This is the reason why a slit aperture does notproduce the same degree of confocal rejection, as does a pinholeaperture. If the fiber bundle is now replaced by an incoherent one, thena random pattern will emerge at plane P (FIG. 1D). Rejection of unwantedlight is more efficient in this case, because the fibers carrying theunwanted photons are now spatially separated, reducing the probabilityfor these photons to pass through the slit. In FIGS. 1A to 1D, theintensity of illumination of each fiber (represented by a circle)corresponds to the darkness of the fiber. The incoherent fiber bundleprovides for an arrangement (position or location) of individual fibersat its output end at D that are scrambled relative to the arrangement ofthe fibers at its input end at P either randomly, or in a prescribedpattern, and as such, the fiber bundle does not preserve an imagecaptured at the input end. However, the location of each fiber of theincoherent fiber bundle at the input and output ends of the fiber may bemapped.

[0017] To form an image, the input illumination line is scanned acrossthe plane P. The scanning can be with any suitable scan mechanism, suchas a galvanometer-mounted mirror. During the scan, the output pattern atplane D is modulated in a random but deterministic fashion. On the wayback, fibers carrying photons from the sample undergo reversetransformation from D to P. In general, what comes out at plane P iscomposed of two components—a line and scattered dots, which originatefrom confocal and unwanted sample planes, respectively. The line movesacross the plane P in synchronism with the input scan line, while thescattered dots blink on and off in a random fashion. The former can bedescanned by the same scanning mirror used for the input, allowing theconfocal (in-focus) component to pass through a stationary slitaperture. The scattered dots are rejected.

[0018] Referring to FIGS. 2A, 2B and 2C, confocal microscopes are shownhaving a probe section with an incoherent fiber optic bundle FB1 and anobjective lens OL and an illuminating and imaging section. Light, suchas produced by a laser, illuminates the incoherence fiber optic bundleFB1 via optics having a first slit aperture S2, and focused by objectivelens OL to the region of interest, such as of tissue. The lightcollected by the lens OL from the region of interest is received by theincoherence fiber optic bundle FB1 and then imaged by onto a detector,such as a CCD camera, via optics having a confocal slit aperture S2.

[0019] The output of the stationary slit aperture rescanned onto a CCDcamera yields a two-dimensional image, as is done in a bilateralscanning slit confocal microscope, in which the CCD camera provideselectronic signals representative of the image to a computer (notshown). The image produced in this way, however, is not an image of thesample but a scrambled image due to the action of the incoherent bundleFB1. The original image can be reconstructed by the computer operatingin accordance with decoding software if the mapping transformations ofeach of the fibers from D to P is known. Alternatively, the output ofthe slit aperture S2 can be rescanned onto an identically mapped“incoherent” bundle FB2, which reverses the scrambling done by the firstbundle FB1 shown in FIG. 2C. The output of the second bundle FB2 canthen be imaged onto a CCD camera. The computer may be coupled to adisplay, as typical of electronic imaging confocal microscopes, to viewimages of the region of interest.

[0020] By way of a specific example of components usable in theillustrated embodiments of the invention, is a 488 mm Ar ion laser(SpectraPhysics Model 2016). The beam expander and collimator expandsand collimates the Ar laser beam to a 3 mm diameter beam by lenses L1and L2 shown in FIG. 2. The beam is then focused by a 75 mm cylindricallens L3 to a line illuminating a slit aperture S1 (25 μm wide by 3 mmlong). A lens pair (L4 and L5) with focal lengths of 75 and 150 mm,respectively, forms a magnified image (50 μm wide by 6 mm long) of theslit at plane P, where one end of an incoherent fiber bundle FB1 islocated. The fiber bundle (Edmund Scientific) FB1 maybe 30 cm long and6.4 mm in diameter, composed of approximately 128×128 fibers withindividual fiber diameter of 50 μm. The other end of the fiber bundleFB1 is placed at the back focal plane (160 mm) of a 100X, 1.25 NA oilimmersion microscope objective OL. Reflections at both ends of the fiberbundle FB1 may be minimized by index-matching to 6 mm thick opticalwindows (not shown). The objective lens OL images individual fibers downto 0.5 μm at the sample. Reflected light from the sample is collected bythe objective lens OL and coupled back into the fiber bundle FB1.

[0021] The optics of the specific embodiments are specified in Tables 1,2 and 3 for the FIG. 2A, 2B and 2C embodiments respectively. The term“f.l.” represents focal length. Note the scanning mirror M1 having frontand back sides M1* and M1**, respectively, may be driven by a galvodriver, or provided by two mirrors at M1* and M1** driven synchronously,such as by a common galvo driver. TABLE 1 Excitation beam path L1: 50 mmf.l. L2: 150 mm f.l. L3: cylindrical lens 75 mm f.l. S1: slit aperture,25 μm wide × 3 mm long L4: 75 mm f.l. BS: beam splitter (dichroic ifused in fluorescence) M1*: front side of bilateral scanning mirror L5:150 mm f.l. W1 & W2: glass windows index matched to fiber bundle (notneeded for fluorescence) OL: objective lens Return beam path OL:objective lens W1 & W2: glass windows index matched to fiber bundle (notneeded for fluorescence) L5: 150 mm f.l. M1*: front side of bilateralscanning mirror BS: beam splitter (dichroic if used in fluorescence) L6:75 mm f.l. S2: slit aperture, 25 μm wide × 3 mm long L7: 75 mm f.l.M1**: back side of bilateral scanning mirror L8: 150 mm f.l.

[0022] TABLE 2 Excitation beam path L1: 50 mm f.l. L2: 150 mm f.l. L3:cylindrical lens 75 mm f.l. S1: slit aperture, 25 μm wide × 3 mm longL4: 75 mm f.l. BS: beam splitter (dichroic if used in fluorescence) M1*:front side of bilateral scanning mirror L5: 150 mm f.l. W1 & W2: glasswindows index matched to fiber bundle (not needed for fluorescence) OL:objective lens Return beam path OL: objective lens W1 & W2: glasswindows index matched to fiber bundle (not needed for fluorescence) L5:150 mm f.l. M1*: front side of bilateral scanning mirror BS: beamsplitter (dichroic if used in fluorescence) L6: 75 mm f.l. S2: slitaperture, 25 μm wide × 3 mm long DET: linear detector array or line scancamera

[0023] TABLE 3 Excitation beam path L1: 50 mm f.l. L2: 150 mm f.l. L3:cylindrical lens 75 mm f.l. S1: slit aperture, 25 μm wide × 3 mm longL4: 75 mm f.l. BS: beam splitter (dichroic if used in fluorescence) M1*:front side of bilateral scanning mirror L5: 150 mm f.l. W1 & W2: glasswindows index matched to fiber bundle (not needed for fluorescence) FB1:fiber bundle OL: objective lens Return beam path OL: objective lens FB1:fiber bundle W1 & W2: glass windows index matched to fiber bundle (notneeded for fluorescence) L5: 150 mm f.l. M1*: front side of bilateralscanning mirror L6: 75 mm f.l. S2: slit aperture, 25 μm wide × 3 mm longL7: 75 mm f.l. M1**: back side of bilateral scanning mirror L8: 150 mmf.l. FB2: identically mapped fiber bundle as FB1

[0024] With a line input at P, the pattern of illuminated fibers at D isobserved with a CCD camera. FIG. 3 shows an exemplary image of the P endof the fiber from a mirror at the position of a sample. Note that thisline pattern may be color codes with colors corresponding to films inthe bundle. Alternatively, the code may be a binary code of on and offillumination regions corresponding to the fibers in location. FIG. 3A isan image of the reflected light when the sample mirror was in focus. Asshown, most of the reflected light is carried by fibers, whichreassemble into the original slit pattern. FIG. 3B is an image at planeP of reflected light when the sample mirror was moved out of focus by ˜1μm. The brightness of the slit is greatly suppressed, while more photonsemerge from the dispersed random fibers. Some of the scattered dots thatappear outside the line in FIG. 3A are due to slight axial positionvariability. Some of the scattered dots can arise because cross talkbetween two adjacent fibers near the D end will appear as separate,scattered pixels at the P end, but cross-talk is also reduced at thecamera, by the slit.

[0025] A rectangular mask over the central column of fibers -digitallyintegrates the brightness values of the pixels captured by the CCDcamera in order to provide the optical sectioning effect with a confocalslit. To show operation of the microscope, the procedure was carried outfor a series of images taken as the sample mirror was stepped throughthe focus in ˜1 μm increments. For comparison, a data set of theintegrated intensities over the entire fiber bundle was taken tosimulate the “wide field” case. A plot of the two data sets,individually normalized with respect to their peak values, is shown inFIG. 4 as a function of mirror position. The FWHM (full width at halfmaximum) of the axial response function is ˜11 μm without the slitaperture and ˜2 μm with the slit aperture.

[0026] The incoherent fiber bundle used in the example given above isonly approximately random in that groups of nearby fibers tend to stayclustered together from one end to the other end of the bundle.Nonetheless, substantial improvement in optical sectioning is achievedwith a slit aperture, as shown in FIG. 4. A bundle that scrambles in apre-set pattern may be preferred. Then software decoding of the imagedoes not require measurement of the fiber mapping. Such a pattern may beone that maps every row of a square matrix into a maximally separatedsquare grid that fills a matrix of the same dimension. In terms of lightbudget, a fiber bundle with a high fill factor and low numericalaperture may be preferable. The low numerical aperture minimizes lightloss due to overfilling of the microscope objective entrance pupil.

[0027] Variations and modifications in the above-described exemplarysystem, within the scope of the invention will become apparent to thoseskilled in this art. Thus, the description should not be taken in alimiting sense.

1. A confocal microscope comprising: at least one probe sectioninsertable into a body for illuminating a region of interest thereof; animaging section generating illumination light, and constructing imagesfrom light remitted from the region of interest; and at least oneflexible incoherent optical coupling element for transmission of lightbetween the imaging section and the probe section, whereby the confocalmicroscope is a remote probe for confocal imaging of tissue at locationswithin the body in place of an endoscope.
 2. The microscope according toclaim 1 wherein said element is an incoherent fiber optic bundle.
 3. Themicroscope according to claim 2 wherein said imaging section comprises aline scanning means which scans across a proximal end of the element. 4.The microscope according to claim 3 further comprising a slit aperturedisposed in the path of light scanned by said means across said proximalend.
 5. The microscope according to claim 2 further comprising anobjective lens at a distal end of said element for focusing a laser beamin said region.
 6. A confocal microscope comprising: at least one probesection insertable into a body having an objective lens; a lightmanipulation section; and at least one fiber bundle coupling between thelight manipulation section and the objective lens, wherein the fiberbundle scrambles light incident to said fiber bundles, whereby theconfocal microscope is a remote probe for confocal imaging of tissue atlocations within the body in place of an endoscope.
 7. The microscopeaccording to claim 6 wherein said fiber bundle has two ends and saidmicroscope further comprises a confocal mask at one of said ends nearsaid manipulating section of the fiber bundle to enhance confocality. 8.The microscope according to claim 6 wherein the fiber bundle is notcoherent in that spatial individual fibers at one of said ends of thebundle are scrambled relative to that at the other of said ends.
 9. Themicroscope according to claim 8 wherein said individual fibers arescrambled randomly.
 10. The microscope according to claim 8 wherein saidindividual fibers are scrambled in a prescribed pattern.
 11. Themicroscope according to claim 6 wherein the incident light forms a line.12. The microscope according to claim 7 wherein said confocal mask is aslit.
 13. The microscope according to claim 6 wherein said fiber bundlehas a distal end and light from the distal end of the fiber bundle isimaged by said objective lens onto a sample, and remitted light from thesample is collected by said objective lens and coupled back into thefiber bundle.
 14. The microscope according to claim 6 in which each endof the fiber bundle is index matched via a window material to reducereflection from fiber ends.
 15. A method for decoding a scrambled imageformed by an incoherent fiber bundle in a microscope insertable into abody comprising the steps of: raster scanning a focused light spot ontoa first end of the fiber bundle; sequentially reading out acorresponding fiber at a second end of said bundle; and constructing amap of the first and second ends, whereby an image formed by lightremitted into the second end can be unscrambled by the mappedrelationship of the first and second ends.
 16. (Canceled)
 17. A methodfor decoding the a scrambled image formed by an incoherent fiber bundlein a microscope insertable into a body comprising the steps of:illuminating a first end of the incoherent fiber bundle with a codedline pattern; imaging corresponding fibers at a second end of saidbundle; and mapping of the first and second ends, whereby an imageformed by light remitted into the second end can be unscrambled by amapped relationship of the first and second ends.
 18. The methodaccording to claim 17 wherein said spatially coded pattern is a binarymasked pattern.
 19. (Canceled)
 20. The method according to claim 17,wherein the coded line pattern is spatially coded.
 21. The methodaccording to claim 17, wherein the coded line pattern is color-coded.22. A confocal microscope comprising: at least one probe sectioninsertable into a subject for illuminating a region of interest thereof;an imaging section generating illumination light, and constructingimages from light remitted from the region of interest; and at least oneflexible incoherent optical coupling element for transmission of lightbetween the imaging section and the probe section, whereby the confocalmicroscope is a remote probe for confocal imaging of tissue at locationswithin the subject in place of an endoscope.
 23. The microscopeaccording to claim 22 wherein said element is an incoherent fiber opticbundle.
 24. The microscope according to claim 23 wherein said imagingsection comprises a line scanning means which scans across a proximalend of the element.
 25. The microscope according to claim 24 furthercomprising a slit aperture disposed in the path of light scanned by saidmeans across said proximal end.
 26. The microscope according to claim 23further comprising an objective lens at a distal end of said element forfocusing a laser beam in said region.
 27. A confocal microscopecomprising: at least one probe section insertable into a subject havingan objective lens; a light manipulation section; and at least one fiberbundle coupling between the light manipulation section and the objectivelens, wherein the fiber bundle scrambles light incident to said fiberbundle, whereby the confocal microscope is a remote probe for confocalimaging of tissue at locations within the subject in place of anendoscope.
 28. The microscope according to claim 27 wherein said fiberbundle has two ends and said microscope further comprises a confocalmask at one of said ends near said manipulating section of the fiberbundle to enhance confocality.
 29. The microscope according to claim 27wherein the fiber bundle is not coherent in that spatial individualfibers at one of said ends of the bundle are scrambled relative to thatat the other of said ends.
 30. The microscope according to claim 29wherein said individual fibers are scrambled randomly.
 31. Themicroscope according to claim 29 wherein said individual fibers arescrambled in a prescribed pattern.
 32. The microscope according to claim27 wherein the incident light forms a line.
 33. The microscope accordingto claim 28 wherein said confocal mask is a slit.
 34. The microscopeaccording to claim 27 wherein said fiber bundle has a distal end andlight from the distal end of the fiber bundle is imaged by saidobjective lens onto a sample, and remitted light from the sample iscollected by said objective lens and coupled back into the fiber bundle.35. The microscope according to claim 27 in which each end of the fiberbundle is index matched via a window material to reduce reflection fromfiber ends.
 36. A method for decoding a scrambled image formed by anincoherent fiber bundle in a microscope insertable into a subjectcomprising the steps of: raster scanning a focused light spot onto afirst end of the fiber bundle; sequentially reading out a correspondingfiber at a second end of said bundle; and constructing a map of thefirst and second ends, whereby an image formed by light remitted intothe second end can be unscrambled by the mapped relationship of thefirst and second ends.
 37. A method for decoding a scrambled imageformed by an incoherent fiber bundle in a microscope insertable into asubject comprising the steps of: illuminating a first end of theincoherent fiber bundle with a coded line pattern; imaging correspondingfibers at a second end of said bundle; and mapping of the first andsecond ends, whereby an image formed by light remitted into the secondend can be unscrambled by a mapped relationship of the first and secondends.
 38. The method according to claim 37 wherein said spatially codedpattern is a binary masked pattern.
 39. The method according to claim37, wherein the coded line pattern is spatially coded.
 40. The methodaccording to claim 37, wherein the coded line pattern is color-coded.